Fluorescent protein development 1) We are studying photoswitching behaviors of photoswitchable fluorescent proteins and their use in Frster Resonance Energy Transfer (FRET) experiments. This includes an ongoing project to develop photoswitchable yellow fluorescent proteins, photoswitchable red fluorescent proteins, and far-red fluorescent proteins for use in conventional diffraction-limited microscopy as well as super-resolution molecular localization microscopy. 2) We collaborate with Joy Zhao and Peter Schuck on their development of new fluorescence ultracentrifugation techniques. We are surveying numerous fluorescent proteins to better define their oligomerization characteristics. Literature surveys have suggested these characteristics have not been rigorously determined and that unforeseen oligomerization leads to aberrant fluorescent protein behavior. 3) We have an ongoing project to develop improved red fluorescent proteins. Current variants display low fluorescence, slow maturation, and/or oligomerization. Biochemical analyses of wild type proteins coupled with site-directed mutagenesis has led to our discoveries of mRuby variants with much decreased self-association, increased brightness, and faster maturation. Cell biology projects 1) Gaetan Herbomel, a former post-doctoral fellow in the lab of Lawrence Tabak, has started work in my lab and is completing a project to image the localization of Golgi apparatus enzymes using Stimulated Emission Depletion (STED) microscopy, Multifocal Structured Illumination Microscopy (MSIM), Photoactivated Localization Microscopy (PALM), and Stochastic Optical Reconstruction Microscopy (STORM). The multi-color super-resolution experiments have required development new data analyses which we have now implemented. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. 2) We collaborate with Jim Kochenderfer (NCI) to study the self-association properties of chimeric antigen receptor (CAR) molecules using our FRET techniques. Instrumentation and imaging development 1) Frster Resonance Energy Transfer (FRET) is a powerful approach to study the interactions of fluorescent molecules. We are developing new approaches which can be performed on a conventional widefield or confocal microscope to image FRET between a donor photoswitchable fluorescent protein, Dronpa, and an acceptor based on a conventional fluorescent protein. The technique which we call photoswitching FRET (psFRET) is based on Dronpas photoswitching properties in the presence and absence of an acceptor. This technique is currently being used to monitor interactions between a number of proteins within living cells. 2) Another FRET based project using psFPs derived directly from the psFRET studies involves imaging homo-FRET by monitoring the anisotropy of the PS-FPs during the photoswitching process as a read-out of protein-protein interactions. This technique, which we called photoswitching anisotropy FRET (psAFRET), differs from measurements of hetero-FRET (energy transfer between two different color proteins) since it monitors energy transfer between copies of the same probe. 3) We are able to use a previously described coordinate-based co-localization (CBC) analysis of PALM/STORM data to develop a plugin which runs within the freely available ImageJ application. It is designed to run with one thread per computer CPU, which drastically improves the computation time. 4) We have also developed a series of image analysis macros that can be run within the ImageJ application to analyze psFRET data. To make this technique more widely useful we have developed an ImageJ plugin that is much more versatile and can be used on almost any dataset for the steps outlined above. In parallel, a second ImageJ plugin was developed to analyze the exponential decay in fluorescence at each pixel as the photoswitchable fluorescent protein switches off. These are available on the ImageJ update site (https://imagej.net/User:Pattersg). 5) We collaborate with the Hari Shroff lab on the development of improved forms of TIRF microscopy. Recent efforts by his lab have resulted in improvements to lateral super-resolution levels while maintain the acquisition speeds necessary for live cell imaging. The emphasis for our collaboration is now to improve axial resolution in this technique.